The Ras-superfamily consists of over 150 signaling molecules that are categorized into several subfamilies based upon sequence homology. The Ras, Rho, Ran, Rab, Arf, and Rem/Rad family is made up of monomeric GTP hydrolyzing proteins (GTPases or G-proteins) of about 21 KDa that interact with and activate multiple downstream effector proteins, resulting in a variety of biological phenomena (Ehrhardt et al., Exp. Hematol., 2002, 30:1089-106; Vojtek et al., J. Biol. Chem., 1998, 273:19925-8; Shields et al., Trends Cell Biol., 2000, 10:147-54; Reuther et al., Curr. Opin. Cell Biol., 2000, 12:157-65). Similar to their large heterotrimeric G-protein α-subunit counterparts, small GTPases act as binary switches that cycle between active (GTP-bound) and inactive (GDP-bound) states. The GTP/GDP cycling of Ras superfamily GTPases is highly regulated by classes of proteins specific to each subfamily. Guanine nucleotide exchange factors (GEFs) activate GTPases by allowing GDP to become displaced by GTP. GTPase activating proteins (GAPs) cause inactivation by accelerating the intrinsic GTPase activity (thus, GTP hydrolysis) of G-proteins leading to an accumulation of inactive GDP-bound protein. Small GTPases can additionally be regulated by guanine nucleotide dissociation inhibitors (GDIs) that prevent nucleotide exchange, sequester GTPases, and block associated downstream signaling.
Members of the Ras and Rho subfamilies are the most extensively studied group of small GTPases and are essential components of the mitogenic signal transduction pathway. Extracellular stimulation of receptor tyrosine kinases, G-protein coupled receptors, or integrins can result in activation of Ras and the prototypical Rho subfamily members RhoA, Rac1, and Cdc42 (Etienne-Manneville et al., Nature, 2002, 420:629-35; Hall, Science, 1998, 279:509-14). Upstream activation of receptors such as G-protein coupled receptors, integrins, or receptor tyrosine kinases lead to RhoGEF activation at the plasma membrane. Once activated, these GTPases further propagate external signals by activating a multitude of downstream effector proteins, resulting in a diversity of cellular responses.
For example, RhoGEFs engage inactive GDP-bound Rho GTPases and facilitate their activation by catalyzing the exchange of GTP for GDP. GTP-bound Rho GTPases take on an active conformation that allows for binding of effectors and further propagation of downstream signaling pathways. The assay of the invention identifies compounds that interfere with the rate-limiting step of Rho GTPase activation; the rate limiting step being guanine nucleotide exchange by RhoGEFs. This assay can be used with virtually any Ras superfamily GTPase and its cognate GEF.
Additionally, activation of Ras and Rho family GTPases is a critical step during tumor progression and acquisition of an invasive and metastatic phenotype (Frame et al., Curr. Opin. Genet. Dev., 2002, 12:36-43; Evers et al., Eur. J. Cancer, 2000, 36:1269-74; Boettner et al., Gene, 2002, 286:155-74; Sahai et al., Nat. Rev. Cancer, 2002, 2:133-42; Oxford et al., Cancer Lett., 2003, 189:117-28). These GTPases are highly oncogenic, with over 30% of all human cancers and 90% of pancreatic cancers harboring activated Ras mutations (Oxford et al., Cancer Lett., 2003, 189:117-28). Additional evidence also suggests a vital role for Rho family members during transformation and the acquisition of an invasive and metastatic phenotype by regulating the actin cytoskeleton (Fritz et al., Br. J. Cancer, 2002, 87:635-44; Fritz et al., Int. J. Cancer, 1999, 81:682-7; Clark et al., Nature, 2000, 406:532-5; Kleer et al., Am. J. Pathol., 2002, 160:579-84). Furthermore, GEFs specific to Rho GTPases (RhoGEFs), such as Dbl (diffuse B cell lymphoma), Tiam1 (T-cell invasion and metastasis factor), and LARG (leukemia associated RhoGEF), are routinely isolated during screens for transforming oncogenes and make up one of the largest classes of human proto-oncogenes, with over 60 members (Whitehead et al., Biochim. Biophys. Acta, 1997, 1332:F1-23; Schmidt et al., Genes Dev., 2002, 16:1587-609).
Further, evidence indicates that the GTPase proteins are also ideal targets for the treatment of neurological damage stemming from spinal cord injury and stroke, as well as neurological disorders such as Alzheimer's disease and Parkinson's disease. Even in the presence of inhibitory substrates inhibition of Rho has been shown to stimulate neurite outgrowth. (Winton et al, J. Biol. Chem., 2002, 277:32820-9). Moreover, inhibition of Rho using C3-toxin-like peptides facilitates the repair of damaged spinal cords in mouse models. However, these Rho antagonists are covalent modifiers and are not inhibitors of guanine nucleotide exchange.
Due to their inherent oncogenic signaling properties and role in cancer metastasis Ras and Rho-mediated signaling events are emerging targets for anti-cancer drug discovery (Downward, Nat. Rev. Cancer, 2003, 3:11-22). Furthermore, the complex pathways that regulate Ras superfamily GTPases are the current focus of intensive research. For many of these pursuits there is a critical need for sensitive, real-time measurements of GTPase activation and subsequent monitoring of signaling events.